AICAR attenuates postoperative abdominal adhesion formation by inhibiting oxidative stress and promoting mesothelial cell repair

Background Postoperative abdominal adhesion is one of most common complications after abdominal operations. 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) is an adenosine 5’-monophosphate activated protein kinase (AMPK) pathway agonist that inhibits inflammation, reduces cell fibrosis and cellular reactive oxygen species (ROS) injury, promotes autophagy and mitochondrial function. This study aimed to explore the mechanism of AICAR in inhibiting adhesion formation. Materials and methods Forty rats were randomly divided into five groups. All of the rats except the sham group received cecal abrasion to establish an adhesion model. The rats in the sodium hyaluronate group were treated with 2 mL sodium hyaluronate before closing the peritoneal cavity. The AICAR 1 and 2 groups were treated with 100 mg/kg and 200 mg/kg AICAR, respectively. Seven days after the operation, all of the rats were euthanized, and the adhesion condition was evaluated by Nair’s system. Inflammation was assessed by Eosin-hematoxylin (HE) staining and transforming growth factor-β (TGF-β1) detection. Oxidative stress effect was determined by ROS, nitric oxide (NO) level, superoxide dismutase (SOD), catalase, glutathione peroxidase (Gpx) and malondialdehyde (MDA) levels in adhesion tissue. Then, Sirius red picric acid staining was used to detect the fiber thickness. Immunohistochemical staining of cytokeratin-19 (CK-19), alpha-smooth muscle actin (α-SMA) and nuclear factor erythroid 2-related factor 2 (Nrf2) was also performed. Finally, HMrSV5 cells were treated with TGF-β1 and AICAR, the mRNA expression of E-cadherin, α-SMA and vimentin was assessed by q-PCR and cellular immunofluorescent staining. Results The rats in the AICAR-treated group had fewer adhesion formation incidences and a reduced Nair’s score. The inflammation was determined by HE staining and TGF-β1 concentration. The ROS, SOD, Catalase, Gpx, MDA levels and fiber thickness were decreased by AICAR treatments compared to the control. However, the NO production, Nrf2 levels and peritoneal mesothelial cell integrity were promoted after AICAR treatments. In vitro work, AICAR treatments reduced E-cadherin, α-SMA and vimentin mRNA level compared to that in the TGF-β1 group. Conclusion AICAR can inhibit postoperative adhesion formation by reducing inflammation, decreasing oxidative stress response and promoting peritoneal mesothelial cell repair.


Introduction
As a most commonly complication after surgery, postoperative abdominal adhesion occured in 90%-95% of patients who underwent abdominal surgery will have abdominal adhesion formation, and 20% of these patients will suffer adhesion-related disease [1]. Despite surgical removal of the adhesion bands, there is currently no other effective treatment [2]. Therefore, preventing postoperative adhesion formation is very important.
The formation of postoperative adhesion is a complex process [3,4]. After surgery, the injury or trauma of peritoneal tissue will lead to inflammation and collagen formation or deposition. Oxidative stress injury plays a vital role, and overaccumulation of superoxide not only harms mesothelial cells but also promotes cell transformation into mesenchymal cells [5]. Inhibiting reactive oxygen species (ROS) is a promising way to prevent adhesion formation [6]. Adenosine 5'-monophosphate activated protein kinase (AMPK) is an important pathway that maintains energy homeostasis and inhibits tissue injury [7]. A previous study illustrated that the AMPK pathway has a protective effect in chronic inflammatory diseases and can inhibit cell fibrosis, reduce ROS injury, promote autophagy, and improve mitochondrial function [8][9][10]. However, the function of the AMPK pathway in abdominal formation has not yet been determined.
The formation of abdominal adhesion is a process of inflammation that in turn leads to collagen deposition or fiber formation; thus, the AMPK pathway may have a protective effect by preventing adhesion in postoperative abdominal adhesion. Here, we demonstrated that the AMPK agonist 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) [11] can reduce adhesion formation by inhibiting oxidative stress and promoting peritoneal repair in a rat model.

Materials and methods
The rats were purchased from the Experimental Animal Center of Xi'an Jiaotong University and kept in an approximately 22±2˚C environment. All rats had free access to food and water. The experiment was approved by the Ethics Committee of Xi'an Jiaotong University (No. XJTULAC2021-318).

Abdominal adhesion animal model
Forty Sprague-Dawley (SD) rats, aged>3months and weighing 200g-250g, were used to establish the abdominal adhesion animal model as described in a our previous study. One day before surgery, the abdominal hair was removed, and fasting water was provided 6 hours before surgery. The rats were anesthetized under isoflurane anesthesia (2% isoflurane with 0.4 L/min O 2 flow). After skin sterilization, a 2-to 3-centimeter incision was created in the middle of the abdomen. Both the right side and cecum were rubbed with dry gauze 30 times until needle-like bleeding points appeared. Then, the injured cecum side was placed on the opposite side of the injured abdominal wall. Finally, the abdominal wall was closed by an intermittent suture in two layers.

Study design and adhesion assessment
Forty rats were randomly divided into five groups. The rats in the sham group received only open and close abdominal wall surgery, and the rats in the other groups underwent the abdominal adhesion model procedure. For the sodium hyaluronate group, 2 mL sodium hyaluronate was spread on the injured side of the abdominal wall and cecum before closing the abdominal wall. Within 7 days after surgery, the control group was treated with 1 mL normal saline through intraperitoneal injection on the left upper side of the abdomen, while AICAR Group 1 and Group 2 were treated with AICAR (#9944p, cell signaling technology, USA, purity>99%) 100 mg/kg and 200 mg/kg via subcutaneous injection, respectively, every day after surgery. Seven days after surgery, the rats were killed, and the adhesion condition was classified accoding to the Nair's score system, described as follows: Grade 0: complete absence of adhesions; Grade 1: single band of adhesion between viscera or from one viscera to the abdominal wall; Grade 2: two bands either between viscera or from viscera to the abdominal wall; Grade 3: more than two bands between viscera or from viscera to the abdominal wall, Grade 4: multiple dense adhesions or viscera directly adherent to the abdominal wall, irrespective of number or extent of adhesive bands [12]. Then, the adherent tissue was collected and divided into two pieces, one kept at -80˚C and the other fixed with 4% paraformaldehyde.

Hematoxylin-eosin (HE) staining and inflammation evaluation
For HE staining, the tissues were first fixed in 4% paraformaldehyde overnight and then made into 4 μm thick serial paraffin sections. HE staining was performed according to instructions of the HE staining kit (Beijing Soleibao Technology Co., Ltd., Beijing, China). The inflammation histopathological score was evaluated by the following system: 0 for the tissue without inflammatory cells; 1 for cases with observable macrophages, lymphocytes, and plasma cells; 2 for cases with macrophages, plasma cells, eosinophils, and neutrophils; and 3 for cases with inflammatory cell infiltration and microabscess formation. This process was performed by two independent researchers. At least five randomly selected high-power fields were reviewed for each section, at least four sections per rat were evaluated, and the score per section was the mean score of all sections per rat.

ELISA
An enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of TGF-β1 production (CSB-E04727r, Shanghai Ximei Chemical Co. Ltd., Shanghai, China) and NO production (EMSNO, Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturers' manual.

Chemiluminescence test for ROS
ROS were measured in the peritoneal lavage fluid 7 days after the operation. The lavage fluids were collected before opening the abdominal cavity. A commercial ROS detection kit (Sinovac Biochemical Reagents, Shanghai, China) was used according to the manufacturer's protocol. The concentrations of the samples were calculated using a standard curve.

Malondialdehyde (MDA) detection
The MDA concentration in the tissues was measured by an MDA detection kit (S0131S, beyotime biotechnology Co. Ltd., Shanghai, China) according to the manufacturer's instructions.

Superoxide dismutase (SOD), Catalase and Glutathione peroxidase (Gpx) activity detection
SOD activity was measured spectrophotometrically according to the methods of Flohe and Otting [13]. In this method, inhibition of the cytochrome c reduction rate is monitored at 550nm at 25˚C utilizing the xantine/xantine oxidase system as the source of O 2 . SOD competes for superoxide and decreases the reduction rate of cytochrome c. One unit of SOD activity was defifined as the amount of enzyme that inhibits by 50% the rate of cytochrome c reduction. The method of Aebi [14] was used to analyze catalase activity. This method uses change in absorbance at 240nm at 25˚C of the solution of 10mM H 2 O 2 in phosphate buffer, pH 7.0. The decrease in absorbance per unit time is a measure of the catalase activity. Gpx activity was assayed by the method of Paglia and Valentine [15]. The rate of oxidation of the reduced form of glutathione was monitored by a decrease in the concentration of NADPH caused by addition of glutathione reductase to the reaction mixture. All enzyme activities were reported as units/mg of protein.

Immunohistochemical (IHC) Staining
IHC staining was used to assess mesothelial cell healing by cytokeratin-19 (CK-19), alphasmooth muscle actin (α-SMA) and nuclear factor erythroid 2-related factor 2 (Nrf2). IHC staining was performed using a streptavidin-biotin kit (Maxim, Fuzhou, China) following the manufacturer's instructions. The sections were deparaffinized and rehydrated, incubated with 30 g/L hydrogen peroxide solution at room temperature for 5 minutes, and blocked with goat serum. Subsequently, the sections were incubated with mouse CK-19 (CK-19; 1 : 50 dilution, Abcam, UK), rabbit α-SMA (ab5694, 1 : 100 dilution, Abcam) and Nrf2 antibodies (Nrf2; 1 : 100 dilution, Abcam) at 4˚C overnight. The sections were then incubated with biotinylated rabbit anti-mouse IgG for 20 minutes. Incubation with streptavidin-biotin peroxidase complex at 37˚C was performed for another 20 minutes. The sections were washed in phosphate-buffered saline four times for 5 minutes per wash. Diaminobenzidine tetrahydrochloride was used for visualization, and hematoxylin was used as the counterstain. The sections were dehydrated, mounted, and sealed. To evaluate the expression of these indicators, at least five random highpower fields of adhesion tissue were reviewed for each section as described in the histopathological evaluation. α-SMA staining was used to assess the fibrosis level of tissue in different treatments by using Image-Pro Plus 5.0 software (Leica Qwin. Plus, Leica Microsystem Imaging Solutions Ltd., Cambridge, UK). Then, CK-19 staining was used to assess the rate of the integrity of the mesothelial cells. This rate was calculated by the stained mesothelial cell length in the adhesion tissue divided by the total mesothelial cell length in the selected fields. The Nrf2 scoring system was as follows: 0-no expression, 1-low expression, 2-moderate expression, 3-strong expression, and 4-very strong expression. The expression condition in the adhesive tissue was calculated as the average score of examined sections.

Sirius red picric acid staining
More than 5 pathological sections were randomly selected for Sirius red picric acid staining using the experimental method. Staining was performed with 0.1% Sirius red picric acid (Direct Red 80; Sigma-Aldrich, St. Louis, MO, USA), followed by counterstaining with hematoxylin. Five high-magnification fields were randomly selected from each pathological section to measure the width of collagen tissue by using Image-Pro Plus 5.0 software. The average thickness of the adhesive area of each tissue was considered the thickness of the adhesive area of each tissue.

Cellular immunofluorescent staining
HMrSV5 cells (5×10 6 ) were grown on cover slips. Then, the cells in the AICAR-treated group were cultured in a 1 mg/mL AICAR solution for 2 hours and 5 ng/mL TGF-β1 for 24 hours, while the control group was treated with the same dose of PBS. Next, after washing three times, the cells were fixed with 4% paraformaldehyde overnight. Cellular immunofluorescent staining was performed as described in previous studies. The fluorescence intensity was measured by ImageJ software and compared to the control group.

RNA isolation and real-time PCR
Total RNA was extracted and purified from cells by a kit according to the manufacturer's instructions (Pioneer Biological Co., Ltd. Xi'an, China). Then, the total RNA was reverse-transcribed using SYBR Green (Takara Biotechnology Co., Ltd.) and an ABI 7500 instrument (Thermo Fisher Scientific, Inc.). Primers were designed according to the sequences reported below ( Table 1). The real-time PCR data were normalized to endogenous levels and were carried out in triplicate.

Statistical analyses
All data were analyzed with SPSS 22.0 software (Chicago, IL, USA) and are presented as the means ± standard errors of the mean (SEM). Analysis of variance (ANOVA) was performed to determine significant differences in normally distributed data, and the abnormally distributed data were analyzed by the Kruskal-Wallis test. Enumeration data were determined by Fisher's exact test. p values <0.05 were considered significant.

AICAR ameliorates adhesion scores in a rat model
All forty rats finished this experiment, and none died or suffered serious postoperative complications. On the 7 th day after surgery, the adhesion scores of all the rats were assessed by Nair's system. All rats in the control group had formed a large number of dense adhesions, while those in the sodium hyaluronate-treated group showed formation of a few thin adhesions. The AICAR-treated group had fewer adhesion formation incidences than the control group ( Fig  1A). The adhesion score, which was evaluated by Nair's scoring system, demonstrated that AICAR treatment attenuated the adhesion score compared to that of the control group. However, the nonadhesion rate was not different from that of the control group (Fig 1B and 1C).

AICAR reduces inflammation in adhesion tissue
Inflammation is the initiating cause of adhesion formation. By the HE staining inflammation scoring system, we found that the inflammation score in the AICAR-treated group was dramatically reduced (Fig 2A and 2B). Next, to explore the AICAR-related inflammation in adhension tissue, the level of key proteins involved in inflammation were measured using ELISA and western-blotting analysis (Fig 2C), TGF-β1 concentration was increased in adhesion tissue, while it was significantly decreased in the AICAR-treated group.

AICAR alleviates the oxidative stress response in adhesion tissue
ROS play a vital role in the inflammatory reaction of the postoperative adhesion formation process. Here, we found that compared to the control group, the AICAR-treated group had a lower level of ROS and a ROS-related index, MDA (p<0.05) (Fig 3A and 3B). However, the NO levels was increased after AICAR treatments (p<0.05) (Fig 3C). The activity of SOD, Catalase and Gpx was also significanty decreased in the AICAR-treated group (p < 0.01) (Fig 3D). Then, we assessed the Nrf2 levels in adhesion tissue and found that AICAR can promote Nrf2 expression in adhesion tissue (p<0.05) (Fig 3E).

AICAR treatment inhibits tissue fibrosis and promotes peritoneal mesothelial cell repair
Adhesion formation is the process of tissue fibrosis. By Sirius red picric acid staining, we found that the AICAR-treated group had a lower fiber thickness than the control group ( Fig  4A1 and 4B). CK19 staining demonstrated that the AICAR groups had a higher rate of peritoneal mesenchymal cell integrity than the control (Fig 4A2 and 4C). α-SMA staining showed that the AICAR-treated groups can significantly reduce the fibrogenesis in vivo (Fig 4A3 and  4D), but the mechanism is still unclear. Peritoneal mesothelial cell mesenchymal transition (MMT) is an important process in fibrosis. Here, in a TGF-β1-induced HMrSV5 human cell MMT model, we found that AICAR treatment can reduced α-SMA and vimentin mRNA expression compared to that in the TGF- β1 group, while the E-cadherin mRNA expression was higher in the AICAR group (Fig 5A-5C). Cell immunofluorescence also showed that the α-SMA fluorescence intensity was downregulated in the AICAR group compared to it in the TGF-β1 group (Fig 5D).

Discussion
The AMPK pathway is an important pathway to maintain energy homeostasis in our body that participates widely in stress (ischemic, oxidative, hypoglycemic), exercise, and hormone (adiponectin, leptin) pathways and can be modulated by pharmacological agents [16]. Here, we demonstrated that AMPK agonists can alleviate inflammatory reactions, decrease oxidative stress response and promote mesothelial cell repair in postoperative adhesion formation. These results demonstrated that promoting the AMPK pathway may prevent adhesion formation.
As an AMPK pathway agonist, AICAR can reduce inflammatory reactions in many disease [17]. AICAR can inhibit NF-kB DNA binding independently of AMPK to attenuate lipopolysaccharide (LPS) triggered inflammatory responses in human macrophages [18]. AICAR can also inhibit NF-kB activation and reduce the expression of interleukin-1β (IL-1β) in inflammatory pain [19]. In an intestinal ischemia and reperfusion model, AICAR treatment has also been shown to decrease the inflammatory response and alleviate tissue damage caused by ischemia [20]. Here, we found by HE staining and TGF-β1 assessment that AICAR can inhibit the inflammation score in adhesion tissue and that the level of inflammatory factor TGF-β1 was also reduced by AICAR treatment.
During the formation of postoperative abdominal adhesions, due to the destruction of local tissues, a large amount of reactive oxygen species is generated, which in turn aggravates the inflammatory response and leads to tissue damage [21]. AICAR can decrease sepsis or LPS-induced endothelial activation and organ injury by reducing oxidative stress response [22]. Here, we found that AICAR may play a citical role in anti-oxidative effects, it can reduce ROS, SOD, catalase and Gpx activity in adhesion tissue. And it can also promote the expression of Nrf2. Nrf2 is best known as an oxidant stress response transcription factor, it lies at the center of a complex regulatory network and has a mutiple function. The most function exerted by Nrf2 is the protective role in anti-inflammation conditions and promoting cells repairment [23]. The peritoneum is composed of peritoneal mesothelial cells and the underlying connective tissue [24]. After damage, peritoneal mesothelial cells are missing. The peritoneal cells that repair the local tissue are derived from the surrounding normal peritoneal cells or other stem cells. However, in an inflammatory environment, these cells can undergo necrosis or become fibroblasts. This transformation will promote the formation of a local adhesion, and thus inhibition of peritoneal mesothelial cell-tointerstitial transformation is very important for the prevention and treatment of postoperative abdominal adhesion [25,26]. In the in vivo experiment, we found that AICAR can reduce collagen deposition and fibrogenesis during adhesion and promote mesothelial cell repair. To further understand its possible mechanism, we extracted and cultured rat primary peritoneal mesothelial cells, and the results showed that AICAR treatment can significantly inhibit TGF-β1-induced peritoneal mesothelial cell-to-mesenchymal transition.
In conclusion, our study demonstrated that an AMPK pathway agonist can inhibit postoperative adhesion formation by reducing inflammation, decreasing oxidative stress response and promoting peritoneal mesothelial cell repair. This finding indicated that AICAR may be used as a potential drug to prevent the formation of postoperative abdominal adhesions.